HIV-1 group M viruses are responsible for the majority of HIV infections worldwide and are genetically classified into nine subtypes . These subtypes display biological differences in pathogenicity and disease progression [2–7], transmissibility [8–10] and treatment outcomes [11–13]. All currently used antiretroviral drugs were developed, evaluated and approved based on their activity and efficacy for HIV-1 subtype B infections, the predominant subtype in the Western Hemisphere and Europe. However, infections by this subtype are a substantial minority of the HIV-1 infections globally. Antiretroviral drug resistance mutation patterns and resistance algorithms are derived from in-vitro and clinical studies of subtype B viruses and infections. The ‘IAS Annual Resistance Mutation Update’  constitutes resistance data from subtype B viruses, whereas resistance mutation data in the ‘HIV Drug Resistance database’ (HIVdb, Stanford University; http://hivdb.stanford.edu/) are inclusive of mutations from various HIV subtypes. We report here on non-nucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutations unique to etravirine and rilpilvirine exposure in subtype B infection occurring in patients with non-B subtypes exposed to nevirapine (NPV) or efavirenz (EFV).
RV288 is a multicenter PEPFAR (President's Emergency Plan For AIDS Relief) Basic Program Evaluation that is being conducted in Kenya, Tanzania, Uganda and Nigeria. This cross-sectional study was developed to estimate the rate of virologic suppression of patients on first-line highly active antiretroviral therapy (HAART) in PEPFAR clinics and to identify predictors of virologic suppression and immunologic recovery. Routine virologic monitoring is not a standard of care in any of these counties. Patients on HAART for at least 6 months were randomly selected for a one-time visit which included viral load (HIV RNA) clinical chemistries and hematology, absolute CD4+ lymphocyte count, and percentage and survey of demographics and behavioral risk factors for transmission. Individuals with an HIV RNA above 1000 copies/ml were referred for intensive adherence counseling while having the sample submitted for automated sequencing to detect antiretroviral drug resistance mutations. HIV RNA was quantified from plasma using Roche Amplicor Version 1.5 with lower detection limit of 400 copies/ml in plasma. Specimens with HIV RNA below 400 copies were rerun on a more sensitive platform (Abbott M2000SP/RT). Genotyping was done on archived plasma samples using the Truegene HIV-1 genotyping kit. The study was approved by the Walter Reed Army Institute of Research Institutional Review Board (IRB) and the Kenya Medical Research Institute IRB.
A total of 975 patients were enrolled comprising individuals from Defense Forces Memorial Hospital (Nairobi, n = 325), Kericho District Hospital (Kericho, n = 136), Kombewa District Hospital (Kombewa, n = 109), Nandi Hills District Hospital (Nandi Hills, n = 97), Kapsabet District Hospital (Kapsabet, n = 111), Kapkatet District Hospital (Kapkatet, n = 110) and Litein Mission Hospital (Litein, n = 87). We found 617 out of 975 patients (63%) had HIV RNA below 400 copies, whereas 215 patients (22%) had HIV RNA above 1000 copies/ml, and samples were submitted for resistance testing. Of the 215 samples submitted for resistance testing, 115 (53%) had one or more resistance mutations. Interestingly, 14 of these patients (13.8%) had an amino acid substitution at reverse transcriptase position 138 (A, G, K or Q; Table 1). These mutations are selected by etravirine and rilpivirine in subtype B viruses, resulting in a reduced susceptibility to these drugs, yet no patient had ever been exposed to either of them. All patients were either on EFV or NVP, which are not reported to produce mutations at this position, at least for subtype B viruses. These 14 reverse transcriptase sequences were analyzed for subtype using the Recombinant Identification Program (RIP) tool (http://www.hiv.lanl.gov/content/sequence/RIP/RIP.html). The sequences were submitted to Genbank and their accession numbers are included in Table 1. It appears as if EFV and NVP can select for mutations at position 138 in non-B subtypes, but less so in subtype B viruses (there is one case with subtype B HIV-1).
Mutations resulting from antiretroviral drug exposure are reported to occur with differing frequencies and display different patterns when subtype B viruses are compared to non-subtype B in the reverse transcriptase, protease and integrase sequences [15–18] Cornelissen et al.  identified natural polymorphisms in non-B subtypes that, although not likely to confer significant resistance alone, could modify the effect of drug-selected mutations influencing the fold-sensitivity phenotype. Non-subtype B viruses have acquired resistance to drugs via mutations that are not selected by those same drugs in subtype B viruses in the HIV-1 protease enzyme [20,21]. A particular mutation or genotypic pattern may display differential drug susceptibility across different subtypes [15,22].
Our findings are unique in that they show NNRTI's EFV and NVP producing a cluster of mutations at position 138 in the reverse transcriptase of subtype A, D and recombinants, which are rarely, if ever, observed in subtype B viruses. The mutations produced characteristically result from exposure to etravirine or rilpivirine, to which none of these patients has ever been exposed. In the Stanford database, under a section designated ‘Mutation prevalence according to subtype and treatment’, the E138A mutation has been observed in patients with subtype A and D infections on EFV or NVP, but not E138G, E138K or E138Q for these subtypes. Additionally, the reported frequency of these mutations is quite a bit lower than what we observe in our sample of patients with subtype A virus .
Patients failing EFV with just K103N mutation, which usually appears first, are likely to achieve complete suppression with etravirine or rilpivirine, allowing the sequencing of agents within the NNRTI class. Etravirine maintains partial activity even in the presence of certain NNRTI mutations commonly produced by NVP and EFV. The potential for patients with non-B infections to acquire E138 mutations from EFV or NVP increases the chances of patients failing these drugs to have cross-resistance with etravirine and rilpivirine, which can occur by other mutations as well (e.g. Y181C,I; M230L) , and emphasizes the importance of resistance testing. Further research is needed to identify factors that may predispose patients on EFV/NVP to acquiring this cluster of mutations.
Conflicts of interest
This research has been supported by the President's Emergency Plan for AIDS Relief (PEPFAR) through the US Department of Defense under the terms of cooperative agreement W81XWH-11-2-0174 with the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. The views expressed are those of the authors and should not be construed to represent the positions of the US Army or DoD.
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